JPS58109480A - Novel physiologically active substance, dihydromonacolin l and its preparation - Google Patents

Abstract

NEW MATERIAL:The novel physiologically active substance, dihydromonacolin L of formula having the following characteristics: neutral and white crysalline powder; soluble in methanol, ethanol, propanol, acetone, ethyl acetate, chloroform and benzene, and insoluble in hexazne and petroleum ether. USE:It has blood cholesterol level depressing activity and is useful as an antilipemic agent, antiarteriosclerotic agent, etc. PROCESS:Dihydromonacoline L-producing microorganism belonging to Monascus genus, e.g. Monascus ruber No.1005 strain (FERM-P No.4882) is cultured aerobically, and the objective dihydromonacolin L is extracted and separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new physiologically active substance having a cholesterol-lowering effect and a method for producing the same.

Hyperlipidemia, particularly hypercholesterolemia, is known to be one of the major causes of heart diseases such as myocardial infarction and arteriosclerosis. Therefore, the present inventor conducted a search for the purpose of discovering an excellent new physiologically active substance having a cholesterol-lowering effect among microbial products.

As a result, from the culture of one strain of mold, the active substance dihuman p.
Monacolin L (DlhydrOmOnaCO■1nL)
was collected.

This substance was found to be effective as a blood cholesterol lowering agent through animal experiments using rats. Furthermore, we investigated the physical and chemical properties of this substance and found that it is a new substance. This substance is hereinafter referred to as dihydromonacorin L.

In the present invention, the following formula (■) is obtained by culturing mold and using the culture.

The present invention relates to a method for collecting dihydromonacolin L represented by the following formula, particularly a method for culturing Monascus genus and collecting dihydromonacolin L from the culture.

The microorganism that can be used in the present invention is a dihydromonacolin L-producing bacterium belonging to the genus Monascus, and a strain that the present inventor recognizes as being particularly effective is, for example, Monascus rubel, /1616 o' 5 strain Thea, and the present strain is It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its accession number is FERM-P.
,% 4822).

Needless to say, any species of the genus Monascus other than those mentioned above may be used, regardless of their variants or mutant strains, as long as they have the ability to produce dihydromonacolin L.

The mycological properties of the dihydromonacolin L-producing bacteria isolated from food products produced in Thailand by the present inventors are as follows.

1) Growth The growth on the valley grass-glucose agar medium (25°C) is fast, and the diameter of one seedling is 5 to 6 cm in 10 days.The colony is flat and relatively thin basal hyphae 4 develop.

Vaporized hyphae are poorly developed, white and mostly woolly. On the basal hyphae I- there are many 5-fruited offspring (C13istotheei).
Forms a) and becomes reddish brown as it matures. Both the front and back sides of the colony are brown to reddish brown.

Growth on Sabouraud agar medium (25°C) is extremely fast, and the colony diameter reaches 6 to 6.5 am' IC in 10 days.

The colony surface is extremely flat, and both basal and vaporized hyphae develop better than on balayage-glucose agar medium. child 5
The number of fruits formed is extremely small. The surface of the colony is reddish-orange to reddish brown, and the underside is reddish brown to dark brown.

Growth on oatmeal agar medium (25°C) 17"l is slow, the diameter of the colony is 15-2 cm in 10 days. The colony is flat, and the development of vaporized mycelia and ascocarp formation are extremely poor. The surface of the colony, Both sides are dark red to reddish brown.

Growth on Zapek agar medium (25°C) is extremely slow, and the diameter of the colony is 16 to 1.8 h in 10 days.

The growth rate at 67°C on the above various agar media is comparable to that at 25"C.

2) Shape The fruit is spherical with a diameter of 30 to 60μ, and the wall of the fruit is thin (,
Membrane. The ascocarp has septa and consists of hyphae with a diameter of 35-4.5μ and a length of 15-80μ. The seedlings have 8 spores, are spherical, and disappear. Ascospores
is oval to oval, 4-5 x 4-7μ, smooth surface. Conidia (Con1dia) are colorless, spherical to pear-shaped, 6 to 9
×6-11μ, the base is cut-like, the wall is relatively thick (smooth surface, basophilically linked. Conidiophores resemble vegetative hyphae, unbranched ~
Branched and forms conidia at the tip. The mycelium is branched, has septa, and most have a diameter of 3 to 5 μm.

As a result of the above observations, this bacterium was found to be Monascus rubel (Mo
nascus ruber (van Tieghem
) ] was identified.

The mycological description of Monascus rubel is detailed in the following paper. Namely, Pan Chigeum: Journal of the French Botanical Society (Bull, Soc, Botan.

France, ) volume 31, page 227, 1884,
Cole et al.: Canadian Journal of Botany
ny) Vol. 46, p. 987, 1968, Taira, Takayama: Bulletin of the Mycological Society of Japan, Vol. 9, p. 128, 1969.

In addition, as mentioned above, this strain is Monascus rubel/M
It has been deposited as strain 1005 at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.

Dihuman p-monafrin L is produced aerobically in a culture using a bacterial strain that produces dihydromonafrin L by a method known as a mold culture method. For example, dihydromonacolin-producing bacteria are subcultured on a medium consisting of 2% soluble starch, 1% glucose, 2% peptone, and 2 ml of agar, and the cells grown on this agar medium are directly transferred to the production medium for dihydromonaco1J7L production. It is also possible to inoculate and culture the cells grown in a production medium, and to culture dihydromonacolin L in a new production medium.

Dihydromonacolin-producing bacteria grow at 7 to 40'G, but a temperature of 20 to 35°C is usually preferred for dihydromonacolin production. For culturing the Monascus bacteria that produces dihydromonacolin L, any nutrient source known for culturing molds and other microorganisms can be used. For example, glucose,
Maltose, dextrin, starch, lactose, saccharose, glycerin, etc. can be used as carbon sources.

Among these carbon sources, glucose and starch are the preferred carbon sources for dihydromonacolin L production.

All known nitrogen sources for the growth of Monascus and other microorganisms can be used to produce dihydromonacolin L. For example, peptone, meat extract, yeast, yeast extract,
Soybean flour, peanut flour, corn flour, rice bran, inorganic nitrogen sources, etc. can be used. When dihydromonacolin L is produced by culturing dihydromonacolin L-producing bacteria, inorganic salts and metal salts are added if necessary. You can also add trace amounts of heavy metals if needed.

Dihydromonacolin can be obtained by aerobically culturing its producing bacteria, and commonly used aerobic culture methods such as solid state culture, shaking culture, and aerated agitation culture methods are used. When defoaming is required during culture or medium sterilization, antifoaming agents such as silicone oil and surfactants can be used. The culture temperature is preferably 20 to 35°C.

The physiological activity of dihydromonacoli/L can be assayed by the following method, which examines its blood cholesterol lowering effect on runt. That is, 5 rats per group were given Triton WR-
1359 (trade name) (this substance has the effect of increasing blood cholesterol levels) 40 omy/Kg was injected intravenously, and at the same time, a fixed dose of dihydromonacolin L was orally administered.
At time 1, the rats were exsanguinated to death and blood cholesterol was measured by a conventional method (administration group). On the other hand, Trydon WR-133
Rats intravenously injected with 9 alone were treated in the same manner and blood cholesterol was measured (control #). By comparing the cholesterol values of both groups, the effectiveness of dihydromonacolin L can be determined definitively. Culture is Nohidrho Monaco IJ 7L
;/l'--Continue to extract the substance from the culture until it is substantially accumulated, depending on the properties of the substance revealed by the inventor, as shown in the Examples below. This can be done by appropriately combining the following methods.

That is, for example, extraction with ether, ethyl acetate, chloroform, benzene, etc., dissolution of highly negative solvents such as 7setone and alcohol, removal of impurities with less polar solvents such as petroleum ether and hexane, and gel f using a Sephadex column. Adsorption chromatography using filtration, activated carbon, silica gel, etc. By using a suitable combination of these means, the substance can be isolated from the culture as a homogeneous substance.

Next, the physical and chemical properties of dihydromonacolin L will be described.

Dihydromonacolino is a white crystalline powder that is soluble in lower alcohols such as methanol, ethanol, propatool, acetone, chloroform, ethyl acetate, benzene, etc., and insoluble in hexane, petroleum ether, etc. The substance is a neutral substance and does not dissolve in neutral or acidic water, but the lactone structure is ring-opened by normal alkali treatment! II Note 9J It converts into a substance and is dissolved in water. p of
Extracted with vinegar, lI ethyl, chloroform, etc. in the H region,
Lydihydromonacolin Lg is reconverted by drying.

The elemental analysis value of dihydromonacolin L is 7450% carbon.
It is 985% hydrogen and 1565% oxygen.

The molecule is t306 and the molecular formula is CI9H2O0g.

Figures 1, 2, and 3 show the optical external absorption spectrum, NMR spectrum, and quality analysis spectrum of book and white matter, respectively.

R
Give a single spon I- to f + r & 0.4 a.

Note that this spot is detected using sulfuric acid spray (color develops reddish brown with mild heating) and iodine.

Acute toxicity of dihydro μ Monacorino L by oral administration in mice <
It has low toxicity with an LD50 of 1 fl/kg or more.

The effect of dihydromonacholine L on lowering blood cholesterol in animals was investigated using various methods, and its usefulness was confirmed.

For example, tryton WR-1339400 for rats.
On November 19th, 100 g of hydromonacholine was administered orally at 1:00 pm, and 14 hours later, the rats were killed by exsanguination and the blood concentration was measured using a conventional method.

As a result, blood cholesterol levels were reduced by 606% when Nohydra Monacolin L was administered compared to when only Triton WR-1339 was intravenously injected.

As mentioned above, dihydromonacolin L has the effect of lowering blood cholesterol levels, such as antilipidemia,
It can be used medicinally as an anti-arteriosclerotic drug.

These compounds can be administered orally or non-injectionally, for example, in capsules, tablets, injections, and the like.

Oral preparations are usually preferred. The dosage varies depending on age, symptoms, body weight, etc., but it is usually about 50 to 50 doses per day for adults.
(It is administered in 1 to 3 divided doses at 1+9'. However, a larger amount can be used if necessary.)

Next, examples of the present invention will be shown. Now that the above-mentioned properties have been clarified by the present invention, based on these findings, it is possible to collect dihydromonacolino L from culture or related sL substances. Various σ) modification means are possible. The present invention is not limited to the examples (but includes all alternatives that can be easily deduced from the findings already described).

Example 1 3% glucose, 1% benoton, 3% defatted soy flour.

Glycerin 7%, NaN05
0. 2%, MgSO4・7H2001
%'a: t b W Monascus loupel hawk 1005 strain was inoculated into the body medium and cultured aerobically at a temperature of 28° C. for 10 days. The obtained culture P solution (50e) was adjusted to pH 3 by adding 6N1 source acid, and then extracted with an equal volume of ethyl acetate.

The extract was concentrated to dryness and dissolved in 1E benzene to remove insoluble materials. P solution is 5% bicarbonate of soda solution 500I7
Washed twice with Il. Then add 0.2 N to the benzene solution.
Add 11 parts of caustic soda solution, stir at room temperature, and add 1 part of benzene.
After confirming by thin layer chromatography that dihydromonacolin L had disappeared from -, the aqueous layer was collected. This aqueous layer was adjusted to pH 3 with 6N salt and extracted twice with 11N ethyl acetate. The extract was concentrated to dryness to obtain 31 g of an oily substance.

Monacolin K is precipitated at the same time as this oil is dissolved in benzene and crystallized. The mother liquor from which the crystals were removed was dried and 80. ! 9 silica gel (Wakogel C-200
) was adsorbed onto the column. Column with dichloromethane 400
rnl, dichloromethane-ethyl acetate (9:1) 51
．． The mixture was developed in the order of 71 dichloromethane-ethyl acetate (8:2). Both parts containing dihydromonacolin L were dried to 40
．． 9 on a column of silica gel (Wako Gel C-200), and added 250 ml of hexane. Hexane-7cetone (
9:1) was developed in the order of 4A'. Dihydromonaphrine L
Dry both parts containing 5% and dissolve in 60ml of benzene
Washed twice with 30 ml of sodium bicarbonate solution. Then, it was dehydrated and concentrated to dryness to obtain 30 m9 of white powder. This white powder was further processed by high-speed liquid polymerization (silica gel).
0DS column, developed with 7 setonitrile/water solvent system)
After collecting the fractions, the solvent was distilled off to obtain dihydromonacolin L18 as a crystalline white powder.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum (KBr) of quahydromonacolin L, and Figure 2 shows the N of dihydro IJ monafrin L.
The MR spectrum (CDC13) and FIG. 3 show the quality 1 analytical spectrum of dihytromona-yl) L, respectively. Patent applicant Akira Endo 1) 11/″ (15)

Claims (1)

[Claims] 1. A new physiologically active substance dihydromonacolin L0 having the following physical and chemical properties 1) Elemental analysis: C74, 50%, H9, 85, Q
15.65% 2) Molecular weight: 506 6) Molecular formula and chemical structure: C+oH+. 034) Infrared absorption spectrum: Shown in FIG. 5) NMR spectrum: shown in FIG. 6) Mass spectrometry spectrum: shown in Figure 3. 7) Soluble in: methanol, ethanol, propatool, 7 setone, ethyl vinegar, chloroform, benzene/hexane. Insoluble in petroleum ether. 8) Material properties and hood: Neutral, white crystalline powder. 2 Dihydromonacolin L (7) # system obtained by culturing dihydro μ monacolin L-producing bacteria belonging to the genus Monascus and isolating dihydromonacolino L. 3. The manufacturing method according to claim 2, wherein the dihydro-7nacoline-producing bacterium belonging to the genus Monascus is Monascus loupel. 4. The production method according to claim 2, wherein the dihydro monacolin L-producing bacterium belonging to the genus Monascus is Nascus loupel/+6,1005a.